PROJECT SUMMARY While most peripheral T-cell lymphomas (PTCLs) carry a grim prognosis, there are several rare subtypes that are particularly lethal, with a median life expectancy of only a few months. We have recently studied three such T-cell lymphomas: hepatosplenic T-cell lymphoma (HSTL), enteropathy-associated T-cell lymphoma (EATL)2 and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL). Although these three diseases are very different in their clinical presentation, pathologic features and risk factors, they are characterized by highly overlapping genetic alterations. Specifically, all three of these tumors have high frequencies of silencing mutations in SETD2 and activating mutations in STAT5B. The mechanisms through which these common mutations lead to very distinct phenotypes are a central theme in this proposal. We hypothesize that HSTL, EATL and MEITL arise from distinct, though currently unknown, cells-of-origin. A common feature of these diseases is that they are predominantly derived from gamma-delta T cells. The role of T-cell receptor signaling in these tumors is poorly understood and will be informed by the work in Project 1. Further, mutations in SETD2 could reveal additional vulnerabilities that will be explored further in Project 3. Defining the normal counterparts of HSTL, EATL, and MEITL may provide a better understanding of the disease biology and the mechanisms of transformation underlying these lethal lymphomas. The advent of single-cell sequencing provides new approaches for defining the molecular profiles of individual cells that give rise to tissues and are resident in different organs. In this proposal, we seek to elucidate the resident T-cell populations of the liver, spleen, Peyer's patch and intestine in mouse and humans to identify different T-cell populations that are present in the tissues from which these three tumors arise. In Aim 1, we will define the cell-of-origin of three lethal T-cell lymphomas: HSTL, EATL and MEITL. These studies will define the nonmalignant cell-of-origin of HSTL, EATL and MEITL through single-cell sequencing approaches and determine the effects of SETD2 and STAT5B in T-cell lineage development. In Aim 2, we will characterize the individual and combined effects of SETD2 and STAT5B mutations in PTCLs. SETD2 is the primary enzyme responsible for trimethylation of lysine 36 on histone H3 (H3K36me3). In the same tumors, we have found STAT5B, a critical component of the JAK-STAT pathway in the T-cell lineage, to be the most frequently activated oncogene across all 3 subtypes. We are developing in vivo mouse models that enable us to study the derangement of these genes in the T-cell lineage. We propose to study the mechanisms of lymphoma initiation and lineage commitment. This work will utilize our extensive experience working with lineage-specific mouse models and characterizing immune cell behavior in normal and malignant cells. Finally, we will define the spectrum of STAT5B and SETD2 alterations across all T-cell lymphomas (with Cores B and C).